Jason Z. Vlahakis

A novel experimental heme oxygenase-1-targeted therapy for hormone-refractory prostate cancer.

Heme oxygenase-1 (HO-1), a member of the heat shock protein family, plays a key role as a sensor and regulator of oxidative stress. Herein, we identify HO-1 as a biomarker and potential therapeutic target for advanced prostate cancer (PCA). Immunohistochemical analysis of prostate tissue using a progression tissue microarray from patients with localized PCA and across several stages of disease progression revealed a significant elevation of HO-1 expression in cancer epithelial cells, but not in surrounding stromal cells, from hormone-refractory PCA (HRPCA) compared with hormone-responsive PCA and benign tissue. Silencing the ho-1 gene in HRPCA cells decreased the HO-1 activity, oxidative stress, and activation of the mitogen-activated protein kinase-extracellular signal-regulated kinase/p38 kinase. This coincided with reduced cell proliferation, cell survival, and cell invasion in vitro, as well as inhibition of prostate tumor growth and lymph node and lung metastases in vivo. The effect of ho-1 silencing on these oncogenic features was mimicked by exposure of cells to a novel selective small-molecule HO-1 inhibitor referred to as OB-24. OB-24 selectively inhibited HO-1 activity in PCA cells, which correlated with a reduction of protein carbonylation and reactive oxygen species formation. Moreover, OB-24 significantly inhibited cell proliferation in vitro and tumor growth and lymph node/lung metastases in vivo. A potent synergistic activity was observed when OB-24 was combined with Taxol. Together, these results establish HO-1 as a potential therapeutic target for advanced PCA.

Selectivity of imidazole–dioxolane compounds for in vitro inhibition of microsomal haem oxygenase isoforms

Haem oxygenases (HO) are involved in the catalytic breakdown of haem to generate carbon monoxide (CO), iron and biliverdin. It is widely accepted that products of haem catabolism are involved in biological signaling in many physiological processes. Conclusions to most studies in this field have gained support from the judicious use of synthetic metalloporphyrins such as chromium mesoporphyrin (CrMP) to selectively inhibit HO. However, metalloporphyrins have also been found to inhibit other haem‐dependent enzymes, such as nitric oxide synthase (NOS), cytochromes P‐450 (CYPs) and soluble guanylyl cyclase (sGC), induce the expression of HO‐1 or exhibit varied toxic effects. To obviate some of these problems, we have been examining non‐porphyrin HO inhibitors and the present study describes imidazole–dioxolane compounds with high selectivity for inhibition of HO‐1 (rat spleen microsomes) compared to HO‐2 (rat brain microsomes) in vitro. (2R,4R)‐2‐[2‐(4‐chlorophenyl)ethyl]‐2‐[(1H‐imidazol‐1‐yl)methyl]‐4‐methyl‐1,3‐dioxolane hydrochloride) was identified as the most selective inhibitor with a concentration of 0.6 μM inhibiting HO‐1(inducible) by 50% compared with 394 μM for HO‐2 (constitutive). These compounds were found to have no effects on the catalytic activities of rat brain NOS and lung sGC, but were potent inhibitors of microsomal CYP2E1 and CYP3A1/3A2 activities. In conclusion, we have identified imidazole–dioxolanes that are able to inhibit microsomal HO in vitro with high selectivity for HO‐1 compared to HO‐2, and little or no effect on the activities of neuronal NOS and sGC. These molecules could be used to facilitate studies on the elucidation of physiological roles of HO/CO in biological systems.

Anti-Plasmodium activity of imidazolium and triazolium salts

We have previously reported that tetrazolium salts were both potent and specific inhibitors of Plasmodium replication, and that they appear to interact with a parasite component that is both essential and conserved. The use of tetrazolium salts in vivo is limited by the potential reduction of the tetrazolium ring to form an inactive, neutral acyclic formazan. To address this issue imidazolium and triazolium salts were synthesized and evaluated as Plasmodium inhibitors. Many of the imidazolium and triazolium salts were highly potent with active concentrations in the nanomolar range in Plasmodium falciparum cultures, and specific to Plasmodium with highly favorable therapeutic ratios. The results corroborate our hypothesis that an electron-deficient core is required so that the compound may thereby interact with a negatively charged moiety on the parasite merozoite; the side groups in the compound then form favorable interactions with adjacent parasite components and thereby determine both the potency and selectivity of the compound.

UDP-Gal: GlcNAc-R β1,4-galactosyltransferase—a target enzyme for drug design. Acceptor specificity and inhibition of the enzyme

Galactosyltransferases are important enzymes for the extension of the glycan chains of glycoproteins and glycolipids, and play critical roles in cell surface functions and in the immune system. In this work, the acceptor specificity and several inhibitors of bovine β1,4-Gal-transferase T1 (β4GalT, EC 2.4.1.90) were studied. Series of analogs of N-acetylglucosamine (GlcNAc) and GlcNAc-carrying glycopeptides were synthesized as acceptor substrates. Modifications were made at the 3-, 4- and 6-positions of the sugar ring of the acceptor, in the nature of the glycosidic linkage, in the aglycone moiety and in the 2-acetamido group. The acceptor specificity studies showed that the 4-hydroxyl group of the sugar ring was essential for β4GalT activity, but that the 3-hydroxyl could be replaced by an electronegative group. Compounds having the anomeric β-configuration were more active than those having the α-configuration, and O-, S- and C-glycosyl compounds were all active as substrates. The aglycone was a major determinant for the rate of Gal-transfer. Derivatives containing a 2-naphthyl aglycone were inactive as substrates although quinolinyl groups supported activity. Several compounds having a bicyclic structure as the aglycone were found to bind to the enzyme and inhibited the transfer of Gal to control substrates. The best small hydrophobic GlcNAc-analog inhibitor was found to be 1-thio-N-butyrylGlcNβ-(2-naphthyl) with a Ki of 0.01 mM. These studies help to delineate β4GalT–substrate interactions and will aid in the development of biologically applicable inhibitors of the enzyme.

X-ray crystal structure of human heme oxygenase-1 in complex with 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethanone: a common binding mode for imidazole-based heme oxygenase-1 inhibitors.

Development of inhibitors specific for heme oxygenases (HOs) should aid our understanding of the HO system and facilitate future therapeutic applications. The crystal structure of human HO-1 complexed with 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethanone (3) was determined. This inhibitor binds to the HO-1 distal pocket such that the imidazolyl moiety coordinates with heme iron while the adamantyl group is stabilized by a hydrophobic binding pocket. Distal helix flexibility, coupled with shifts in proximal residues and heme, acts to expand the distal pocket, thus accommodating the bulky inhibitor without displacing heme. Inhibitor binding effectively displaces the catalytically critical distal water ligand. Comparison with the binding of 2-[2-(4-chlorophenyl)ethyl]-2-[1H-imidazol-1-yl)methyl]-1,3-dioxolane (2) revealed a common binding mode, despite differing chemical structures beyond the imidazolyl moiety. The inhibitor binding pocket is flexible, yet contains well-defined subpockets to accommodate appropriate functional groups. On the basis of these structural insights, we rationalize binding features to optimize inhibitor design.

The anti-malarial activity of bivalent imidazolium salts.

A series of compounds containing bivalent imidazolium rings and one triazolium analog were synthesized and evaluated for their ability to inhibit the replication of Plasmodium falciparum cultures. The activity and selectivity of the compounds for P. falciparum cultures were found to depend on the presence of electron-deficient rings that were spaced an appropriate distance apart. The activity of the compounds was not critically dependent on the nature of the linker between the electron-deficient rings, an observation that suggests that the rings were responsible for the primary interaction with the molecular target of the compounds in the parasite. The bivalent imidazolium and triazolium compounds disrupted the process whereby merozoites gain entry into erythrocytes, however, they did not appear to prevent merozoites from forming. The compounds were also found to be active in a murine Plasmodium berghei infection, a result consistent with the compounds specifically interacting with a parasite component that is required for replication and is conserved between two Plasmodium species.

Inhibition of the Enzymatic Activity of Heme Oxygenases by Azole-Based Antifungal Drugs

Ketoconazole (KTZ) and other azole antifungal agents are known to have a variety of actions beyond the inhibition of sterol synthesis in fungi. These drugs share structural features with a series of novel heme oxygenase (HO) inhibitors designed in our laboratory. Accordingly, we hypothesized that therapeutically used azole-based antifungal drugs are effective HO inhibitors. Using gas chromatography to quantify carbon monoxide formation in vitro and in vivo, we have shown that azole-containing antifungal drugs are potent HO inhibitors. Terconazole, sulconazole, and KTZ were the most potent drugs with IC50 values of 0.41 ± 0.01, 1.1 ± 0.4, and 0.3 ± 0.1 μM for rat spleen microsomal HO activity, respectively. Kinetic characterization revealed that KTZ was a noncompetitive HO inhibitor. In the presence of KTZ (2.5 and 10 μM), Km values for both rat spleen and brain microsomal HO were not altered; however, a significant decrease in the catalytic capacity (Vmax) was observed (P < 0.005). KTZ was also found to weakly inhibit nitric-oxide synthase with an IC50 of 177 ± 2 μM but had no effect on the enzymatic activity of NADPH cytochrome P450 reductase. Because these drugs were effective within the concentration range observed in humans, it is possible that inhibition of HO may play a role in some of the pharmacological actions of these antimycotic drugs.

Neurotherapeutic effects of novel HO‐1 inhibitors in vitro and in a transgenic mouse model of Alzheimer's disease

Heme oxygenase‐1 (HO‐1) encoded by the HMOX1 gene is a 32‐kDa stress protein that catabolizes heme to biliverdin, free iron, and carbon monoxide (CO). Glial HO‐1 is over‐expressed in the CNS of subjects with Alzheimers disease (AD), Parkinsons disease (PD), and multiple sclerosis (MS). The HMOX1 gene is exquisitely sensitive to oxidative stress and is induced in brain and other tissues in various models of disease and trauma. Induction of the glial HMOX1 gene may lead to pathological brain iron deposition, intracellular oxidative damage, and bioenergetic failure in AD and other human CNS disorders such as PD and MS. Therefore, targeted suppression of glial HO‐1 hyperactivity may prove to be a rational and effective therapeutic intervention in AD and related neurodegenerative disorders. In this study, we report the effects of QC‐47, QC‐56, and OB‐28, novel azole‐based competitive and reversible inhibitors of HO‐1, on oxidative damage to whole‐cell and mitochondrial compartments in rat astrocytes transfected with the HMOX1 gene. We also report the effect of OB‐28 on the behavior and neuropathology of APPswe/PS1∆E9 mice. OB‐28 was found to reduce oxidative damage to whole‐cell and mitochondrial compartments in rat astrocytes transfected with the HMOX1 gene. Moreover, OB‐28 was found to significantly counter behavioral deficits and neuropathological alterations in APPswe/PS1∆E9 mice. Attenuation of AD‐associated behavioral deficits and neuropathological changes suggests that HO‐1 may be a promising target for neuroprotective intervention in AD and other neurodegenerative diseases.

Effectiveness of Novel Imidazole-Dioxolane Heme Oxygenase Inhibitors in Renal Proximal Tubule Epithelial Cells

To enhance our understanding of the physiological roles of heme oxygenase (HO) isozymes, HO-1 (inducible) and HO-2 (constitutive), we developed novel imidazole-based HO inhibitors. Unlike the metalloporphyrins, these imidazole-dioxolane compounds are selective for the in vitro inhibition of HO with minimal effects on other heme-dependent enzymes such as nitric oxide synthase and soluble guanylyl cyclase. In the current study, we tested the hypothesis that these novel HO inhibitors are effective in intact cells by extending their application to cultured, renal proximal tubule epithelial cells (LLC-PK1). HO-1 and HO-2 protein expression was enhanced by pretreatment of cells with hemin, transduction with adenovirus encoding human HO-1, and transfection with cDNA for HO-2, respectively. Total HO activity was measured by determining the formation of carbon monoxide (CO), whereas cell viability and apoptosis were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the expression of activated caspase-3. Gliotoxin/tumor necrosis factor-α (TNF-α) produced cytotoxicity in wild-type LLC-PK1 cells (P < 0.05) but not in HO-1 and HO-2 overexpressing or wild type cells pretreated with hemin (10 μM). The presence of imidazole-dioxolane HO inhibitors (2–25 μM) decreased cell viability (P < 0.05). A CO-releasing molecule reversed, in a dose-dependent manner, the cytotoxic effects and caspase-3 activation induced by the combination of gliotoxin/TNF-α and the HO inhibitors, suggesting an important role for CO in protection against renal toxicity. These data demonstrate a protective role of both HO-1 and HO-2 against gliotoxin/TNF-α-induced cytotoxicity in LLC-PK1 cells. The novel imidazole-dioxolane compounds can be used as effective inhibitors of HO activity in cell culture.

Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN. In spite of cloning many O-antigen genes, biochemical characterization has been done on very few enzymes involved in O-antigen synthesis. In this work, we expressed the wbdN gene in E. coli BL21, and the His-tagged protein was purified. WbdN activity was characterized using the donor substrate UDP-[(14)C]Glc and the synthetic acceptor substrate GalNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. The enzyme product was isolated by high pressure liquid chromatography, and mass spectrometry showed that one Glc residue was transferred to the acceptor by WbdN. Nuclear magnetic resonance analysis of the product structure indicated that Glc was β1-3 linked to GalNAc. WbdN contains a conserved DxD motif and requires divalent metal ions for full activity. WbdN activity has an optimal pH between 7 and 8 and is highly specific for UDP-Glc as the donor substrate. GalNAcα derivatives lacking the diphosphate group were inactive as substrates, and the enzyme did not transfer Glc to GlcNAcα-O-PO(3)-PO(3)-(CH(2))(11)-O-Ph. Our results illustrate that WbdN is a specific UDP-Glc:GalNAcα-diphosphate-lipid β1,3-Glc-transferase. The enzyme is a target for the development of inhibitors to block O157-antigen synthesis.